Methods and compositions for diagnosis and prognosis of appendicitis and differentiation of causes of abdominal pain

ABSTRACT

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in appendicitis patients and in patients at risk for appendicitis. In particular, the invention relates to using as-says that detect one or more biomarkers as diagnostic and prognostic biomarker assays in such patients.

The present application claims priority from U.S. Provisional PatentApplication 61/880,765 filed Sep. 20, 2013, which is hereby incorporatedin its entirety including all tables, figures and claims.

BACKGROUND OF THE INVENTION

The following discussion of the background of the invention is merelyprovided to aid the reader in understanding the invention and is notadmitted to describe or constitute prior art to the present invention.

Acute appendicitis is an inflammatory condition which typically resultsfrom a primary obstruction of the appendix lumen. Once obstructed, theappendix subsequently swells, increasing pressures within the lumen andthe walls of the appendix, resulting in thrombosis and occlusion of thesmall vessels, and stasis of lymphatic flow. The causative agents ofappendicitis include foreign bodies, trauma, intestinal worms,lymphadenitis, and, most commonly, calcified fecal deposits known asappendicoliths or fecaliths. Diagnosis is based on patient history,symptoms and physical examination. Typical appendicitis usually includesabdominal pain beginning in the region of the umbilicus for severalhours, associated with anorexia, nausea or vomiting. The pain typicallysettles into the right lower quadrant.

A commonly used acronym for diagnosis is PALF: pain, anorexia,leukocytosis, and fever. Atypical histories lack this typicalprogression and may include pain in the right lower quadrant as aninitial symptom. Atypical histories often require imaging withultrasound and/or CT scanning. Blood tests for appendicitis are normal,and so not diagnostic, in about 50% of cases. These tests tend to berelatively simple. An abnormal rise in the number of white blood cellsin the blood is a crude indicator of infection or inflammation going onin the body. Such a rise is not specific to appendicitis alone. If it isabnormally elevated, with a good history and examination findingspointing towards appendicitis, the likelihood of having the disease ishigher. Imaging tests such as CT, while useful, expose the recipient todiagnostic levels of radiation.

In terms of biomarkers, C-reactive protein (CRP), an acute-phaseresponse protein produced by the liver in response to inflammatoryprocesses, as been used by clinicians Likewise, other generalinflammatory markers such as procalcitonin, Interleukin-6 (IL-6),Interleukin-8 (IL-8), high mobility group box-1 protein (HMGB1)S100A8/A9, etc., have been studied by clinicians. Like the number ofwhite blood cells, however, these are not specific biomarkers ofappendicitis, and so exhibit poor specificity in use. Leucine-richalpha-2-glycoprotein (LRG) was recently suggested to be a more specificindicator of appendicitis in a pediatric population. Kentsis et al.,Ann. Emerg. Med. 55: 62-70.e4. Epub 2009 Jun. 25; Kharbanda et al.,Academic Emerg. Med. 19: 56-62, 2012.

There remains a need in the art for a rapid, objective, clinicallyaccurate, available diagnostic tool for aiding in the diagnosis and careof appendicitis.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to the identification and use ofdiagnostic markers for appendicitis. The methods and compositionsdescribed herein can meet the need in the art for rapid, sensitive andspecific diagnostic assay to be used in the diagnosis anddifferentiation of abdominal pain and the identification ofappendicitis. In various aspects, the invention relates to materials andprocedures for identifying markers that are associated with thediagnosis, prognosis, or differentiation of appendicitis in a patient;to using such markers in diagnosing and treating a patient and/or tomonitor the course of a treatment regimen; to using such markers toidentify subjects at risk for one or more adverse outcomes related toappendicitis; and for screening compounds and pharmaceuticalcompositions that might provide a benefit in treating or preventing suchconditions.

In a first aspect, the invention discloses methods for determining adiagnosis or prognosis related to appendicits, or for differentiatingbetween causes of abdominal pain. As described herein, measurement ofone or more biomarkers selected from the group consisting of 72 kDa typeIV collagenase, Adiponectin, Advanced glycosylation end product-specificreceptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein,Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase,Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonicanhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5,C—C motif chemokine 1, C—C motif chemokine 13, C—C motif chemokine 15,C—C motif chemokine 17, C—C motif chemokine 19, C—C motif chemokine 20,C—C motif chemokine 21, C—C motif chemokine 22, C—C motif chemokine 23,C—C motif chemokine 24, C—C motif chemokine 26, C—C motif chemokine 27,C—C motif chemokine 3, C—C motif chemokine 4, C—C motif chemokine 7, C—Cmotif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta,Collagenase 3, C-Peptide, Creatine Kinase-MB, C—X—C motif chemokine 10,C—X—C motif chemokine 11, C—X—C motif chemokine 13, C—X—C motifchemokine 16, C—X—C motif chemokine 5, C—X—C motif chemokine 6, C—X—Cmotif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin,Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastricinhibitory polypeptide, Glucagon, Glucagon-like peptide 1,Granulocyte-macrophage colony-stimulating factor, Growth-regulatedalpha, beta, and gamma proteins, Heparin-binding growth factor 2,Hepatocyte growth factor, Immunoglobulin A, Immunoglobulin M,Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, ImmunoglogulinG4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-likegrowth factor-binding protein 2, Insulin-like growth factor-bindingprotein 3, Insulin-like growth factor-binding protein 4, Insulin-likegrowth factor-binding protein 5, Insulin-like growth factor-bindingprotein 6, Insulin-like growth factor-binding protein 7, Interferonalpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta,Interleukin-1 receptor antagonist protein, Interleukin-1 receptor typeI, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12,Interleukin-12 subunit beta, Interleukin-13, Interleukin-15,Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23,Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33,Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha,Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9,Interstitial collagenase, Islet amyloid polypeptide, Keratin, type Icytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitoryfactor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulatingfactor 1, Macrophage metalloelastase, Macrophage migration inhibitoryfactor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinaseinhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growthfactor, Pro-interleukin-16, Prolactin, Prostate-specific antigen,Protein S100-A12, Protransforming growth factor alpha, Secretoryimmunoglobulin A, Serum amyloid P-component, SL cytokine, Stromalcell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromallymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligandsuperfamily member 10, Tumor necrosis factor ligand superfamily member6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosisfactor receptor superfamily member 1B, Vascular endothelial growthfactor receptor 1, Vascular endothelial growth factor receptor 2,Vascular endothelial growth factor receptor 3, von Willebrand Factor,and WAP four-disulfide core domain protein 2 (each referred to hereinfor convenience as an “appendicitis biomarker”) can be used fordiagnosis, prognosis, risk stratification, monitoring, categorizing anddetermination of further diagnosis and treatment regimens in patientshaving or suspected of having appendicitis. The appendicitis biomarkersof the present invention may be used, individually or in panelscomprising a plurality of appendicitis biomarkers. The presence oramount of such marker(s) in a sample obtained from the subject can beused to rule in or rule out appendicitis, and to monitor subjects forimproving or worsening conditions related to appendicitis.

In a first aspect, the present invention relates to methods forevaluating an appendicitis patient or a patient being evaluated for apossible diagnosis. These methods comprise performing an assay methodthat is configured to detect one or more biomarkers selected from thegroup consisting of 72 kDa type IV collagenase, Adiponectin, Advancedglycosylation end product-specific receptor, Alpha-2 macroglobulin,Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1,Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, CancerAntigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-relatedcell adhesion molecule 5, C—C motif chemokine 1, C—C motif chemokine 13,C—C motif chemokine 15, C—C motif chemokine 17, C—C motif chemokine 19,C—C motif chemokine 20, C—C motif chemokine 21, C—C motif chemokine 22,C—C motif chemokine 23, C—C motif chemokine 24, C—C motif chemokine 26,C—C motif chemokine 27, C—C motif chemokine 3, C—C motif chemokine 4,C—C motif chemokine 7, C—C motif chemokine 8, Ceruloplasmin,Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, CreatineKinase-MB, C—X—C motif chemokine 10, C—X—C motif chemokine 11, C—X—Cmotif chemokine 13, C—X—C motif chemokine 16, C—X—C motif chemokine 5,C—X—C motif chemokine 6, C—X—C motif chemokine 9, Cystatin-C,Endothelial protein C receptor, Eotaxin, Epidermal growth factorreceptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide,Glucagon, Glucagon-like peptide 1, Granulocyte-macrophagecolony-stimulating factor, Growth-regulated alpha, beta, and gammaproteins, Heparin-binding growth factor 2, Hepatocyte growth factor,Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, ImmunoglogulinG2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growthfactor-binding protein 1, Insulin-like growth factor-binding protein 2,Insulin-like growth factor-binding protein 3, Insulin-like growthfactor-binding protein 4, Insulin-like growth factor-binding protein 5,Insulin-like growth factor-binding protein 6, Insulin-like growthfactor-binding protein 7, Interferon alpha-2, Interferon gamma,Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptorantagonist protein, Interleukin-1 receptor type I, Interleukin-1receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunitbeta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20,Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29,Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5,Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunitbeta, Interleukin-7, Interleukin-9, Interstitial collagenase, Isletamyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kitligand, Leptin, Leukemia inhibitory factor, Lymphotactin,Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophagemetalloelastase, Macrophage migration inhibitory factor, Matrilysin,Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2,Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin,Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor,Pro-interleukin-16, Prolactin, Prostate-specific antigen, ProteinS100-A12, Protransforming growth factor alpha, Secretory immunoglobulinA, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumornecrosis factor, Tumor necrosis factor ligand superfamily member 10,Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factorreceptor superfamily member 1A, Tumor necrosis factor receptorsuperfamily member 1B, Vascular endothelial growth factor receptor 1,Vascular endothelial growth factor receptor 2, Vascular endothelialgrowth factor receptor 3, von Willebrand Factor, and WAP four-disulfidecore domain protein 2, the results of which assay(s) is/are thencorrelated to the status of the patient. This correlation to status mayinclude one or more of the following: diagnosis of acute appendicitis;indication of a prognosis resulting from acute appendicitis. Forconvenience, patients being evaluated in this manner are referred toherein as “appendicitis patients,” whether or not the appendicitisdiagnosis has been confirmed at the time of the evaluation.

In certain embodiments, the methods for evaluating a patient describedherein are methods for risk stratification of the patient; that is,assigning a likelihood of one or more future changes in health status tothe patient. In these embodiments, the assay result(s) is/are correlatedto one or more such future changes. A level or a change in level of oneor more appendicitis biomarkers, which in turn is(are) associated withan increased probability of morbidity or death, is referred to as being“associated with an increased predisposition to an adverse outcome” in apatient.

In such risk stratification embodiments, preferably the likelihood orrisk assigned is that an event of interest is more or less likely tooccur within 180 days of the time at which the body fluid sample isobtained from the appendicitis patient. In particularly preferredembodiments, the likelihood or risk assigned relates to an event ofinterest occurring within a shorter time period such as 18 months, 120days, 90 days, 60 days, 45 days, 30 days, 21 days, 14 days, 7 days, 5days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, 12 hours, orless. A risk at 0 hours of the time at which the body fluid sample isobtained from the appendicitis patient is equivalent to diagnosis of acurrent condition.

For a positive going marker, an increased likelihood of the occurrenceof a diagnosis is assigned to the patient when the measuredconcentration is above the threshold (relative to the likelihoodassigned when the measured concentration is below the threshold);alternatively, when the measured concentration is below the threshold,an increased likelihood of the nonoccurrence of a diagnosis may beassigned to the patient (relative to the likelihood assigned when themeasured concentration is above the threshold). For a negative goingmarker, an increased likelihood of the occurrence of a diagnosis isassigned to the patient when the measured concentration is below thethreshold (relative to the likelihood assigned when the measuredconcentration is above the threshold); alternatively, when the measuredconcentration is above the threshold, an increased likelihood of thenonoccurrence of a diagnosis may be assigned to the patient (relative tothe likelihood assigned when the measured concentration is below thethreshold).

In certain embodiments, a biomarker or panel of biomarkers is correlatedto a condition or disease by merely its presence or absence. In otherembodiments, a threshold level of a diagnostic or prognostic indicatorcan be established, and the level of the indicator in a patient samplecan simply be compared to the threshold level. A variety of methods maybe used by the skilled artisan to arrive at a desired threshold valuefor use in these methods. For example, for a positive going marker thethreshold value may be determined from a population of patients nothaving acute appendicitis by selecting a concentration representing the75^(th), 85^(th), 90^(th), 95^(th), or 99^(th) percentile of anappendicitis biomarker or biomarkers measured in such “normal” patients.Alternatively, the threshold value may be determined from a “diseased”population of patients by selecting a concentration representing the75^(th), 85^(th), 90^(th), 95^(th), or 99^(th) percentile of a biomarkeror biomarkers measured in patients suffering from acute appendicitis.

Alternatively, the threshold value may be determined from a “diseased”population of appendicitis patients having a predisposition for anoutcome such as death, worsening disease, etc.), by selecting aconcentration representing the 75^(th), 85^(th), 90^(th), 95^(th), or99^(th) percentile of a biomarker or biomarkers measured in patientssuffering from acute appendicitis and who later suffered from theoutcome of interest.

In another alternative, the threshold value may be determined from aprior measurement of a biomarker or biomarkers in the same patient; thatis, a temporal change in the level of a biomarker or biomarkers in thesame patient may be used to assign a diagnosis or a prognosis to thepatient. For example, a diagnostic indicator may be determined at aninitial time, and again at a second time. In such embodiments, anincrease in the marker from the initial time to the second time may bediagnostic of appendicitis or a given prognosis.

The foregoing discussion is not meant to imply, however, that theappendicitis biomarkers of the present invention must be compared tocorresponding individual thresholds. Methods for combining assay resultscan comprise the use of multivariate logistical regression, loglinearmodeling, neural network analysis, n-of-m analysis, decision treeanalysis, calculating ratios of markers, etc. This list is not meant tobe limiting. In these methods, a composite result which is determined bycombining individual markers may be treated as if it is itself a marker;that is, a threshold may be determined for the composite result asdescribed herein for individual markers, and the composite result for anindividual patient compared to this threshold.

The ability of a particular test to distinguish two populations can beestablished using ROC analysis. For example, ROC curves established froma “first” subpopulation which has a particular disease (or which ispredisposed to some outcome), and a “second” subpopulation which doesnot have the disease (or is not so predisposed) can be used to calculatea ROC curve, and the area under the curve provides a measure of thequality of the test. Preferably, the tests described herein provide aROC curve area greater than 0.5, preferably at least 0.6, morepreferably 0.7, still more preferably at least 0.8, even more preferablyat least 0.9, and most preferably at least 0.95.

In certain aspects, the measured concentration of one or moreappendicitis biomarkers, or a composite of such markers, may be treatedas continuous variables. For example, any particular concentration canbe converted into a corresponding probability of existing disease, of afuture outcome for the appendicitis patient, or mortality, of a SIRSclassification, etc. In yet another alternative, a threshold that canprovide an acceptable level of specificity and sensitivity in separatinga population of appendicitis patients into “bins” such as a “first”subpopulation and a “second” subpopulation. A threshold value isselected to separate this first and second population by one or more ofthe following measures of test accuracy:

an odds ratio greater than 1, preferably at least about 2 or more orabout 0.5 or less, more preferably at least about 3 or more or about0.33 or less, still more preferably at least about 4 or more or about0.25 or less, even more preferably at least about 5 or more or about 0.2or less, and most preferably at least about 10 or more or about 0.1 orless;a specificity of greater than 0.5, preferably at least about 0.6, morepreferably at least about 0.7, still more preferably at least about 0.8,even more preferably at least about 0.9 and most preferably at leastabout 0.95, with a corresponding sensitivity greater than 0.2,preferably greater than about 0.3, more preferably greater than about0.4, still more preferably at least about 0.5, even more preferablyabout 0.6, yet more preferably greater than about 0.7, still morepreferably greater than about 0.8, more preferably greater than about0.9, and most preferably greater than about 0.95;a sensitivity of greater than 0.5, preferably at least about 0.6, morepreferably at least about 0.7, still more preferably at least about 0.8,even more preferably at least about 0.9 and most preferably at leastabout 0.95, with a corresponding specificity greater than 0.2,preferably greater than about 0.3, more preferably greater than about0.4, still more preferably at least about 0.5, even more preferablyabout 0.6, yet more preferably greater than about 0.7, still morepreferably greater than about 0.8, more preferably greater than about0.9, and most preferably greater than about 0.95;at least about 75% sensitivity, combined with at least about 75%specificity;a positive likelihood ratio (calculated as sensitivity/(1-specificity))of greater than 1, at least about 2, more preferably at least about 3,still more preferably at least about 5, and most preferably at leastabout 10; ora negative likelihood ratio (calculated as (1-sensitivity)/specificity)of less than 1, less than or equal to about 0.5, more preferably lessthan or equal to about 0.3, and most preferably less than or equal toabout 0.1.

The term_(—) “about” in the context of any of the above measurementsrefers to +/−5% of a given measurement.

Multiple thresholds may also be used to assess a patient. For example, a“first” subpopulation identified by an existing disease, predispositionto a future outcome for the appendicitis patient, predisposition tomortality, etc., and a “second” subpopulation which is not sopredisposed can be combined into a single group. This group is thensubdivided into three or more equal parts (known as tertiles, quartiles,quintiles, etc., depending on the number of subdivisions). An odds ratiois assigned to appendicitis patients based on which subdivision theyfall into. If one considers a tertile, the lowest or highest tertile canbe used as a reference for comparison of the other subdivisions. Thisreference subdivision is assigned an odds ratio of 1. The second tertileis assigned an odds ratio that is relative to that first tertile. Thatis, someone in the second tertile might be 3 times more likely to sufferone or more future changes in disease status in comparison to someone inthe first tertile. The third tertile is also assigned an odds ratio thatis relative to that first tertile.

In certain embodiments, the assay method is an immunoassay. Antibodiesfor use in such assays will specifically bind a full length appendicitisbiomarker of interest, and may also bind one or more polypeptides thatare “related” thereto, as that term is defined hereinafter. Numerousimmunoassay formats are known to those of skill in the art. Preferredbody fluid samples are selected from the group consisting of urine,blood, serum, saliva, tears, and plasma.

The foregoing method steps should not be interpreted to mean that theappendicitis biomarker assay result(s) is/are used in isolation in themethods described herein. Rather, additional variables or other clinicalindicia may be included in the methods described herein. For example, arisk stratification, diagnostic, classification, monitoring, etc. methodmay combine the assay result(s) with one or more variables measured forthe appendicitis patient selected from the group consisting ofdemographic information (e.g., weight, sex, age, race), clinicalvariables (e.g., blood pressure, temperature, respiration rate), riskscores (Alvarado score, Pediatric Appendicitis Score, etc.). This listis not meant to be limiting.

When more than one marker is measured, the individual markers may bemeasured in samples obtained at the same time, or may be determined fromsamples obtained at different (e.g., an earlier or later) times. Theindividual markers may also be measured on the same or different bodyfluid samples. For example, one appendicitis biomarker may be measuredin a serum or plasma sample and another appendicitis biomarker may bemeasured in a urine sample. In addition, assignment of a likelihood maycombine an individual biomarker assay result with temporal changes inone or more additional variables.

In various related aspects, the present invention also relates todevices and kits for performing the methods described herein. Suitablekits comprise reagents sufficient for performing an assay for at leastone of the described appendicitis biomarkers, together with instructionsfor performing the described threshold comparisons.

In certain embodiments, reagents for performing such assays are providedin an assay device, and such assay devices may be included in such akit. Preferred reagents can comprise one or more solid phase antibodies,the solid phase antibody comprising antibody that detects the intendedbiomarker target(s) bound to a solid support. In the case of sandwichimmunoassays, such reagents can also include one or more detectablylabeled antibodies, the detectably labeled antibody comprising antibodythat detects the intended biomarker target(s) bound to a detectablelabel. Additional optional elements that may be provided as part of anassay device are described hereinafter.

Detectable labels may include molecules that are themselves detectable(e.g., fluorescent moieties, electrochemical labels, ecl(electrochemical luminescence) labels, metal chelates, colloidal metalparticles, etc.) as well as molecules that may be indirectly detected byproduction of a detectable reaction product (e.g., enzymes such ashorseradish peroxidase, alkaline phosphatase, etc.) or through the useof a specific binding molecule which itself may be detectable (e.g., alabeled antibody that binds to the second antibody, biotin, digoxigenin,maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA,dsDNA, etc.).

Generation of a signal from the signal development element can beperformed using various optical, acoustical, and electrochemical methodswell known in the art. Examples of detection modes include fluorescence,radiochemical detection, reflectance, absorbance, amperometry,conductance, impedance, interferometry, ellipsometry, etc. In certain ofthese methods, the solid phase antibody is coupled to a transducer(e.g., a diffraction grating, electrochemical sensor, etc) forgeneration of a signal, while in others, a signal is generated by atransducer that is spatially separate from the solid phase antibody(e.g., a fluorometer that employs an excitation light source and anoptical detector). This list is not meant to be limiting. Antibody-basedbiosensors may also be employed to determine the presence or amount ofanalytes that optionally eliminate the need for a labeled molecule.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and compositions for diagnosis,differential diagnosis, risk stratification, monitoring, classifying anddetermination of treatment regimens in patients diagnosed with, or atrisk of, appendicitis. In various embodiments, a measured concentrationof one or more biomarkers selected from the group consisting of 72 kDatype IV collagenase, Adiponectin, Advanced glycosylation endproduct-specific receptor, Alpha-2 macroglobulin,Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1,Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, CancerAntigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-relatedcell adhesion molecule 5, C—C motif chemokine 1, C—C motif chemokine 13,C—C motif chemokine 15, C—C motif chemokine 17, C—C motif chemokine 19,C—C motif chemokine 20, C—C motif chemokine 21, C—C motif chemokine 22,C—C motif chemokine 23, C—C motif chemokine 24, C—C motif chemokine 26,C—C motif chemokine 27, C—C motif chemokine 3, C—C motif chemokine 4,C—C motif chemokine 7, C—C motif chemokine 8, Ceruloplasmin,Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, CreatineKinase-MB, C—X—C motif chemokine 10, C—X—C motif chemokine 11, C—X—Cmotif chemokine 13, C—X—C motif chemokine 16, C—X—C motif chemokine 5,C—X—C motif chemokine 6, C—X—C motif chemokine 9, Cystatin-C,Endothelial protein C receptor, Eotaxin, Epidermal growth factorreceptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide,Glucagon, Glucagon-like peptide 1, Granulocyte-macrophagecolony-stimulating factor, Growth-regulated alpha, beta, and gammaproteins, Heparin-binding growth factor 2, Hepatocyte growth factor,Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, ImmunoglogulinG2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growthfactor-binding protein 1, Insulin-like growth factor-binding protein 2,Insulin-like growth factor-binding protein 3, Insulin-like growthfactor-binding protein 4, Insulin-like growth factor-binding protein 5,Insulin-like growth factor-binding protein 6, Insulin-like growthfactor-binding protein 7, Interferon alpha-2, Interferon gamma,Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptorantagonist protein, Interleukin-1 receptor type I, Interleukin-1receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunitbeta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20,Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29,Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5,Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunitbeta, Interleukin-7, Interleukin-9, Interstitial collagenase, Isletamyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kitligand, Leptin, Leukemia inhibitory factor, Lymphotactin,Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophagemetalloelastase, Macrophage migration inhibitory factor, Matrilysin,Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2,Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin,Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor,Pro-interleukin-16, Prolactin, Prostate-specific antigen, ProteinS100-A12, Protransforming growth factor alpha, Secretory immunoglobulinA, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumornecrosis factor, Tumor necrosis factor ligand superfamily member 10,Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factorreceptor superfamily member 1A, Tumor necrosis factor receptorsuperfamily member 1B, Vascular endothelial growth factor receptor 1,Vascular endothelial growth factor receptor 2, Vascular endothelialgrowth factor receptor 3, von Willebrand Factor, and WAP four-disulfidecore domain protein 2 or one or more markers related thereto, arecorrelated to the status of the patient. As described herein,measurement of one or more biomarkers of the present invention may beused, individually or in panels comprising a plurality of biomarkers, inmethods and compositions for the diagnosis, prognosis, ordifferentiation of abdominal pain in order to rule in or outappendicitis and/or a particular outcome. Such markers can be used indiagnosing and treating a subject and/or to monitor the course of atreatment regimen; for screening subjects for the occurrence or risk ofa particular disease; and for screening compounds and pharmaceuticalcompositions that might provide a benefit in treating or preventing suchconditions.

For purposes of this document, the following definitions apply:

The term “subject” as used herein refers to a human or non-humanorganism. Thus, the methods and compositions described herein areapplicable to both human and veterinary disease. Further, while asubject is preferably a living organism, the invention described hereinmay be used in post-mortem analysis as well. Preferred subjects arehumans, and most preferably “patients,” which as used herein refers toliving humans that are receiving medical care for a disease orcondition. This includes persons with no defined illness who are beinginvestigated for signs of pathology. An “appendicitis patient” is apatient exhibiting symptoms consistent with appendicitis and beingevaluated for its presence, absence, or outcome

Conditions within the differential diagnosis include gallbladder attack,kidney infection, pneumonia, rheumatic fever, diabetic ketoacidosis,ectopic pregnancy, twisted ovarian cyst, hemorrhaging ovarian follicle,urinary tract infection, ulcerative colitis, pancreatitis, intestinalobstruction, pelvic inflammatory disease, diverticulitis, carcinoma ofthe colon, and aortic aneurysm. In preferred embodiments, the biomarkersof the present invention distinguish appendicitis from one or more ofthese mimicking conditions.

Preferably, an analyte such as an appendicitis biomarker is measured ina sample. Such a sample may be obtained from a patient, such as anappendicitis patient. Preferred samples are body fluid samples.

The term “body fluid sample” as used herein refers to a sample of bodilyfluid obtained for the purpose of diagnosis, prognosis, classificationor evaluation of an appendicitis patient of interest, such as a patientor transplant donor. In certain embodiments, such a sample may beobtained for the purpose of determining the outcome of an ongoingcondition or the effect of a treatment regimen on a condition. Preferredbody fluid samples include blood, serum, plasma, urine, saliva, sputum,and pleural effusions. In addition, one of skill in the art wouldrealize that certain body fluid samples would be more readily analyzedfollowing a fractionation or purification procedure, for example,separation of whole blood into serum or plasma components.

The term “diagnosis” as used herein refers to methods by which theskilled artisan can estimate and/or determine the probability (“alikelihood”) of whether or not a patient is suffering from a givendisease or condition. In the case of the present invention, “diagnosis”includes using the results of an assay, most preferably an immunoassay,for an appendicitis biomarker of the present invention, optionallytogether with other clinical characteristics, to arrive at a diagnosis(that is, the occurrence or nonoccurrence) of a disease or condition.That such a diagnosis is “determined” is not meant to imply that thediagnosis is 100% accurate. Many biomarkers are indicative of multipleconditions. The skilled clinician does not use biomarker results in aninformational vacuum, but rather test results are used together withother clinical indicia to arrive at a diagnosis. Thus, a measuredbiomarker level on one side of a predetermined diagnostic thresholdindicates a greater likelihood of the occurrence of disease in theappendicitis patient relative to a measured level on the other side ofthe predetermined diagnostic threshold.

Similarly, a prognostic risk signals a probability (“a likelihood”) thata given course or outcome will occur. A level or a change in level of aprognostic indicator, which in turn is associated with an increasedprobability of morbidity or mortality is referred to as being“indicative of an increased likelihood” of an adverse outcome in apatient.

As used herein, the term “relating a signal to the presence or amount”of an analyte reflects the following understanding. Assay signals aretypically related to the presence or amount of an analyte through theuse of a standard curve calculated using known concentrations of theanalyte of interest. As the term is used herein, an assay is “configuredto detect” an analyte if an assay can generate a detectable signalindicative of the presence or amount of a physiologically relevantconcentration of the analyte. Because an antibody epitope is on theorder of 8 amino acids, an immunoassay configured to detect a marker ofinterest will also detect polypeptides related to the marker sequence,so long as those polypeptides contain the epitope(s) necessary to bindto the antibody or antibodies used in the assay. The term “relatedmarker” as used herein with regard to a biomarker such as one of theappendicitis biomarkers described herein refers to one or morefragments, variants, etc., of a particular marker or its biosyntheticparent that may be detected as a surrogate for the marker itself or asindependent biomarkers. The term also refers to one or more polypeptidespresent in a biological sample that are derived from the biomarkerprecursor complexed to additional species, such as binding proteins,receptors, heparin, lipids, sugars, etc.

In this regard, the skilled artisan will understand that the signalsobtained from an immunoassay are a direct result of complexes formedbetween one or more antibodies and the target biomolecule (i.e., theanalyte) and polypeptides containing the necessary epitope(s) to whichthe antibodies bind. While such assays may detect the full lengthbiomarker and the assay result be expressed as a concentration of abiomarker of interest, the signal from the assay is actually a result ofall such “immunoreactive” polypeptides present in the sample. Expressionof biomarkers may also be determined by means other than immunoassays,including protein measurements (such as dot blots, western blots,chromatographic methods, mass spectrometry, etc.) and nucleic acidmeasurements (mRNA quatitation). This list is not meant to be limiting.With regard to biomarkers which exist in one form as type-I, type-II, orGPI-anchored membrane proteins, such membrane proteins typicallycomprise a substantial extracellular domain, some or all of which can bedetected as soluble forms present in aqueous samples such as blood,serum, plasma, urine, etc., either as cleavage products or as splicevariants which delete an effective membrane spanning domain. Preferredassays detect soluble forms of these biomarkers.

The term “positive going” marker as that term is used herein refer to amarker that is determined to be elevated in patients suffering from adisease or condition, relative to those not suffering from that diseaseor condition. The term “negative going” marker as that term is usedherein refer to a marker that is determined to be reduced in patientssuffering from a disease or condition, relative to patients notsuffering from that disease or condition.

Appendicitis Biomarkers

The following table provides a list of the biomarkers of the presentinvention, together with the Swiss-Prot entry number for the humanprecursor. As noted above, these biomarkers are referred to forconvenience herein as “appendicitis biomarkers.”

SwissProtNum Preferred Name Q15848 Adiponectin Q15109 Advancedglycosylation end product-specific receptor P01023 Alpha-2 macroglobulinP02765 Alpha-2-HS-glycoprotein P02771 Alpha-fetoprotein Q15389Angiopoietin-1 P03973 Antileukoproteinase P08519 Apolipoprotein(a)P15941 Cancer Antigen 15-3 N/A Cancer Antigen 19-9 Q16790 Carbonicanhydrase 9 P06731 Carcinoembryonic antigen-related cell adhesionmolecule 5 P22362 C-C motif chemokine 1 Q99616 C-C motif chemokine 13Q16663 C-C motif chemokine 15 Q92583 C-C motif chemokine 17 Q99731 C-Cmotif chemokine 19 P78556 C-C motif chemokine 20 O00585 C-C motifchemokine 21 O00626 C-C motif chemokine 22 P55773 C-C motif chemokine 23O00175 C-C motif chemokine 24 Q9Y258 C-C motif chemokine 26 Q9Y4X3 C-Cmotif chemokine 27 P10147 C-C motif chemokine 3 P13236 C-C motifchemokine 4 P80098 C-C motif chemokine 7 P80075 C-C motif chemokine 8P00450 Ceruloplasmin P01233 Choriogonadotropin subunit beta P45452Collagenase 3 P01308 C-Peptide (aa57-87) P12277; Creatine Kinase-MBP06732 P02778 C-X-C motif chemokine 10 O14625 C-X-C motif chemokine 11O43927 C-X-C motif chemokine 13 Q9H2A7 C-X-C motif chemokine 16 P42830C-X-C motif chemokine 5 P80162 C-X-C motif chemokine 6 Q07325 C-X-Cmotif chemokine 9 P01034 Cystatin-C Q9UNN8 Endothelial protein Creceptor P51671 Eotaxin P00533 Epidermal growth factor receptor P02792;Ferritin P02794 P02671; Fibrinogen P02675; P02679 P09681 Gastricinhibitory polypeptide P01275 Glucagon P01275 Glucagon-like peptide 1(aa98-127; aa98-128) P04141 Granulocyte-macrophage colony-stimulatingfactor P09341; Growth-regulated alpha, beta, and gamma proteins P19875;P19876 P09038 Heparin-binding growth factor 2 P14210 Hepatocyte growthfactor N/A Immunoglobulin A n/a Immunoglobulin M n/a Immunoglogulin G1N/A Immunoglogulin G2 N/A Immunoglogulin G3 n/a Immunoglogulin G4 P01308Insulin P08833 Insulin-like growth factor-binding protein 1 P18065Insulin-like growth factor-binding protein 2 P17936 Insulin-like growthfactor-binding protein 3 P22692 Insulin-like growth factor-bindingprotein 4 P24593 Insulin-like growth factor-binding protein 5 P24592Insulin-like growth factor-binding protein 6 Q16270 Insulin-like growthfactor-binding protein 7 P01563 Interferon alpha-2 P01579 Interferongamma P01583 Interleukin-1 alpha P01584 Interleukin-1 beta P18510Interleukin-1 receptor antagonist protein P14778 Interleukin-1 receptortype I P27930 Interleukin-1 receptor type II P20809 Interleukin-11P29459; Interleukin-12 P29460 P29460 Interleukin-12 subunit beta P35225Interleukin-13 P40933 Interleukin-15 P60568 Interleukin-2 Q9NYY1Interleukin-20 Q9HBE4 Interleukin-21 Q9NPF7; Interleukin-23 P29460Q8IZJ0 Interleukin-28A Q8IU54 Interleukin-29 P08700 Interleukin-3 O95760Interleukin-33 P05112 Interleukin-4 P05113 Interleukin-5 P08887Interleukin-6 receptor subunit alpha P40189 Interleukin-6 receptorsubunit beta P13232 Interleukin-7 P15248 Interleukin-9 P03956Interstitial collagenase P10997 Islet amyloid polypeptide P08727Keratin, type I cytoskeletal 19 (aa311-367) P21583 Kit ligand P41159Leptin P15018 Leukemia inhibitory factor P47992 Lymphotactin P01374Lymphotoxin-alpha P09603 Macrophage colony-stimulating factor 1 P39900Macrophage metalloelastase P14174 Macrophage migration inhibitory factorP09237 Matrilysin P01033 Metalloproteinase inhibitor 1 P16035Metalloproteinase inhibitor 2 P35625 Metalloproteinase inhibitor 3Q99727 Metalloproteinase inhibitor 4 P02144 Myoglobin P01298 Pancreaticprohormone P10082 Peptide YY P01133 Pro-epidermal growth factor Q14005Pro-interleukin-16 P01236 Prolactin P07288 Prostate-specific antigenP80511 Protein S100-A12 P01135 Protransforming growth factor alpha N/ASecretory immunoglobulin A P02743 Serum amyloid P-component P49771 SLcytokine P48061 Stromal cell-derived factor 1 P08254 Stromelysin-1P40225 Thrombopoietin Q969D9 Thymic stromal lymphopoietin P01375 Tumornecrosis factor P50591 Tumor necrosis factor ligand superfamily member10 P48023 Tumor necrosis factor ligand superfamily member 6 P19438 Tumornecrosis factor receptor superfamily member 1A P20333 Tumor necrosisfactor receptor superfamily member 1B P17948 Vascular endothelial growthfactor receptor 1 P35968 Vascular endothelial growth factor receptor 2P35916 Vascular endothelial growth factor receptor 3 P04275 vonWillebrand Factor Q14508 WAP four-disulfide core domain protein 2 P05231Interleukin-6 P08253 72 kDa type IV collagenase

Marker Assays

In general, immunoassays involve contacting a sample containing orsuspected of containing a biomarker of interest with at least oneantibody that specifically binds to the biomarker. A signal is thengenerated indicative of the presence or amount of complexes formed bythe binding of polypeptides in the sample to the antibody. The signal isthen related to the presence or amount of the biomarker in the sample.Numerous methods and devices are well known to the skilled artisan forthe detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272;5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press,New York, 1994, each of which is hereby incorporated by reference in itsentirety, including all tables, figures and claims.

The assay devices and methods known in the art can utilize labeledmolecules in various sandwich, competitive, or non-competitive assayformats, to generate a signal that is related to the presence or amountof the biomarker of interest. Suitable assay formats also includechromatographic, mass spectrographic, and protein “blotting” methods.Additionally, certain methods and devices, such as biosensors andoptical immunoassays, may be employed to determine the presence oramount of analytes without the need for a labeled molecule. See, e.g.,U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is herebyincorporated by reference in its entirety, including all tables, figuresand claims. One skilled in the art also recognizes that roboticinstrumentation including but not limited to Beckman ACCESS®, AbbottAXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems are among theimmunoassay analyzers that are capable of performing immunoassays. Butany suitable immunoassay may be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like.

Antibodies or other polypeptides may be immobilized onto a variety ofsolid supports for use in assays. Solid phases that may be used toimmobilize specific binding members include include those developedand/or used as solid phases in solid phase binding assays. Examples ofsuitable solid phases include membrane filters, cellulose-based papers,beads (including polymeric, latex and paramagnetic particles), glass,silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGAgels, SPOCC gels, and multiple-well plates. An assay strip could beprepared by coating the antibody or a plurality of antibodies in anarray on solid support. This strip could then be dipped into the testsample and then processed quickly through washes and detection steps togenerate a measurable signal, such as a colored spot. Antibodies orother polypeptides may be bound to specific zones of assay deviceseither by conjugating directly to an assay device surface, or byindirect binding. In an example of the later case, antibodies or otherpolypeptides may be immobilized on particles or other solid supports,and that solid support immobilized to the device surface.

Biological assays require methods for detection, and one of the mostcommon methods for quantitation of results is to conjugate a detectablelabel to a protein or nucleic acid that has affinity for one of thecomponents in the biological system being studied. Detectable labels mayinclude molecules that are themselves detectable (e.g., fluorescentmoieties, electrochemical labels, metal chelates, etc.) as well asmolecules that may be indirectly detected by production of a detectablereaction product (e.g., enzymes such as horseradish peroxidase, alkalinephosphatase, etc.) or by a specific binding molecule which itself may bedetectable (e.g., biotin, digoxigenin, maltose, oligohistidine,2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and detectable label conjugates oftencomprise the use of chemical cross-linkers. Cross-linking reagentscontain at least two reactive groups, and are divided generally intohomofunctional cross-linkers (containing identical reactive groups) andheterofunctional cross-linkers (containing non-identical reactivegroups). Homobifunctional cross-linkers that couple through amines,sulfhydryls or react non-specifically are available from many commercialsources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyldisulfides are thiol reactive groups. Maleimides, alkyl and arylhalides, and alpha-haloacyls react with sulfhydryls to form thiol etherbonds, while pyridyl disulfides react with sulfhydryls to produce mixeddisulfides. The pyridyl disulfide product is cleavable. Imidoesters arealso very useful for protein-protein cross-links. A variety ofheterobifunctional cross-linkers, each combining different attributesfor successful conjugation, are commercially available.

In certain aspects, the present invention provides kits for the analysisof the described appendicitis biomarkers. The kit comprises reagents forthe analysis of at least one test sample which comprise at least oneantibody that binds an appendicitis biomarker. The kit can also includedevices and instructions for performing one or more of the diagnosticand/or prognostic correlations described herein. Preferred kits willcomprise an antibody pair for performing a sandwich assay, or a labeledspecies for performing a competitive assay, for the analyte. Preferably,an antibody pair comprises a first antibody conjugated to a solid phaseand a second antibody conjugated to a detectable label, wherein each ofthe first and second antibodies bind an appendicitis biomarker. Mostpreferably each of the antibodies are monoclonal antibodies. Theinstructions for use of the kit and performing the correlations can bein the form of labeling, which refers to any written or recordedmaterial that is attached to, or otherwise accompanies a kit at any timeduring its manufacture, transport, sale or use. For example, the termlabeling encompasses advertising leaflets and brochures, packagingmaterials, instructions, audio or video cassettes, computer discs, aswell as writing imprinted directly on kits.

Antibodies

The term “antibody” as used herein refers to a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope. See, e.g.Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y.(1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J.Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

Antibodies used in the immunoassays described herein preferablyspecifically bind to an appendicitis biomarker of the present invention.The term “specifically binds” is not intended to indicate that anantibody binds exclusively to its intended target since, as noted above,an antibody binds to any polypeptide displaying the epitope(s) to whichthe antibody binds. Rather, an antibody “specifically binds” if itsaffinity for its intended target is about 5-fold greater when comparedto its affinity for a non-target molecule which does not display theappropriate epitope(s). Preferably the affinity of the antibody will beat least about 5 fold, preferably 10 fold, more preferably 25-fold, evenmore preferably 50-fold, and most preferably 100-fold or more, greaterfor a target molecule than its affinity for a non-target molecule. Inpreferred embodiments, preferred antibodies bind with affinities of atleast about 10⁷ M⁻¹, and preferably between about 10⁸ M⁻¹ to about 10⁹M⁻¹, about 10⁹ M⁻¹ to about 10¹⁰ M⁻¹, or about 10¹⁰ M⁻¹ to about 10¹²M⁻¹.

Affinity is calculated as K_(d)=k_(off)/k_(on) (k_(off) is thedissociation rate constant, K_(on) is the association rate constant andK_(d) is the equilibrium constant). Affinity can be determined atequilibrium by measuring the fraction bound (r) of labeled ligand atvarious concentrations (c). The data are graphed using the Scatchardequation: r/c=K(n−r): where r=moles of bound ligand/mole of receptor atequilibrium; c=free ligand concentration at equilibrium; K=equilibriumassociation constant; and n=number of ligand binding sites per receptormolecule. By graphical analysis, r/c is plotted on the Y-axis versus ron the X-axis, thus producing a Scatchard plot. Antibody affinitymeasurement by Scatchard analysis is well known in the art. See, e.g.,van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold,Comput. Methods Programs Biomed. 27: 65-8, 1988.

The term “epitope” refers to an antigenic determinant capable ofspecific binding to an antibody. Epitopes usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.Conformational and nonconformational epitopes are distinguished in thatthe binding to the former but not the latter is lost in the presence ofdenaturing solvents.

Numerous publications discuss the use of phage display technology toproduce and screen libraries of polypeptides for binding to a selectedanalyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87,6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith,Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. Abasic concept of phage display methods is the establishment of aphysical association between DNA encoding a polypeptide to be screenedand the polypeptide. This physical association is provided by the phageparticle, which displays a polypeptide as part of a capsid enclosing thephage genome which encodes the polypeptide. The establishment of aphysical association between polypeptides and their genetic materialallows simultaneous mass screening of very large numbers of phagebearing different polypeptides. Phage displaying a polypeptide withaffinity to a target bind to the target and these phage are enriched byaffinity screening to the target. The identity of polypeptides displayedfrom these phage can be determined from their respective genomes. Usingthese methods a polypeptide identified as having a binding affinity fora desired target can then be synthesized in bulk by conventional means.See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in itsentirety, including all tables, figures, and claims.

The antibodies that are generated by these methods may then be selectedby first screening for affinity and specificity with the purifiedpolypeptide of interest and, if required, comparing the results to theaffinity and specificity of the antibodies with polypeptides that aredesired to be excluded from binding. The screening procedure can involveimmobilization of the purified polypeptides in separate wells ofmicrotiter plates. The solution containing a potential antibody orgroups of antibodies is then placed into the respective microtiter wellsand incubated for about 30 min to 2 h. The microtiter wells are thenwashed and a labeled secondary antibody (for example, an anti-mouseantibody conjugated to alkaline phosphatase if the raised antibodies aremouse antibodies) is added to the wells and incubated for about 30 minand then washed. Substrate is added to the wells and a color reactionwill appear where antibody to the immobilized polypeptide(s) arepresent.

The antibodies so identified may then be further analyzed for affinityand specificity in the assay design selected. In the development ofimmunoassays for a target protein, the purified target protein acts as astandard with which to judge the sensitivity and specificity of theimmunoassay using the antibodies that have been selected. Because thebinding affinity of various antibodies may differ; certain antibodypairs (e.g., in sandwich assays) may interfere with one anothersterically, etc., assay performance of an antibody may be a moreimportant measure than absolute affinity and specificity of an antibody.

While the present application describes antibody-based binding assays indetail, alternatives to antibodies as binding species in assays are wellknown in the art. These include receptors for a particular target,aptamers, etc. Aptamers are oligonucleic acid or peptide molecules thatbind to a specific target molecule. Aptamers are usually created byselecting them from a large random sequence pool, but natural aptamersalso exist. High-affinity aptamers containing modified nucleotides canconfer improved characteristics on the ligand, such as improved in vivostability or improved delivery characteristics. Examples of suchmodifications include chemical substitutions at the ribose and/orphosphate and/or base positions, and may include amino acid side chainfunctionalities.

Assay Correlations

The term “correlating” as used herein in reference to the use ofbiomarkers refers to comparing the presence or amount of thebiomarker(s) in a patient to its presence or amount in persons known tosuffer from, or known to be at risk of, a given condition; or in personsknown to be free of a given condition. Often, this takes the form ofcomparing an assay result in the form of a biomarker concentration to apredetermined threshold selected to be indicative of the occurrence ornonoccurrence of a disease or the likelihood of some future outcome.

Selecting a diagnostic threshold involves, among other things,consideration of the probability of disease, distribution of true andfalse diagnoses at different test thresholds, and estimates of theconsequences of treatment (or a failure to treat) based on thediagnosis. For example, when considering administering a specifictherapy which is highly efficacious and has a low level of risk, fewtests are needed because clinicians can accept substantial diagnosticuncertainty. On the other hand, in situations where treatment optionsare less effective and more risky, clinicians often need a higher degreeof diagnostic certainty. Thus, cost/benefit analysis is involved inselecting a diagnostic threshold.

Suitable thresholds may be determined in a variety of ways. For example,one recommended diagnostic threshold for the diagnosis of acutemyocardial infarction using cardiac troponin is the 97.5th percentile ofthe concentration seen in a normal population. Another method may be tolook at serial samples from the same patient, where a prior “baseline”result is used to monitor for temporal changes in a biomarker level.

Population studies may also be used to select a decision threshold.Reciever Operating Characteristic (“ROC”) arose from the field of signaldectection therory developed during World War II for the analysis ofradar images, and ROC analysis is often used to select a threshold ableto best distinguish a “diseased” subpopulation from a “nondiseased”subpopulation. A false positive in this case occurs when the persontests positive, but actually does not have the disease. A falsenegative, on the other hand, occurs when the person tests negative,suggesting they are healthy, when they actually do have the disease. Todraw a ROC curve, the true positive rate (TPR) and false positive rate(FPR) are determined as the decision threshold is varied continuously.Since TPR is equivalent with sensitivity and FPR is equal to1-specificity, the ROC graph is sometimes called the sensitivity vs(1-specificity) plot. A perfect test will have an area under the ROCcurve of 1.0; a random test will have an area of 0.5. A threshold isselected to provide an acceptable level of specificity and sensitivity.

In this context, “diseased” is meant to refer to a population having onecharacteristic (the presence of a disease or condition or the occurrenceof some outcome) and “nondiseased” is meant to refer to a populationlacking the characteristic. While a single decision threshold is thesimplest application of such a method, multiple decision thresholds maybe used. For example, below a first threshold, the absence of diseasemay be assigned with relatively high confidence, and above a secondthreshold the presence of disease may also be assigned with relativelyhigh confidence. Between the two thresholds may be consideredindeterminate. This is meant to be exemplary in nature only.

In addition to threshold comparisons, other methods for correlatingassay results to a patient classification (occurrence or nonoccurrenceof disease, likelihood of an outcome, etc.) include decision trees, rulesets, Bayesian methods, and neural network methods. These methods canproduce probability values representing the degree to which a patientbelongs to one classification out of a plurality of classifications.

Measures of test accuracy may be obtained as described in Fischer etal., Intensive Care Med. 29: 1043-51, 2003, and used to determine theeffectiveness of a given biomarker. These measures include sensitivityand specificity, predictive values, likelihood ratios, diagnostic oddsratios, and ROC curve areas. The area under the curve (“AUC”) of a ROCplot is equal to the probability that a classifier will rank a randomlychosen positive instance higher than a randomly chosen negative one. Thearea under the ROC curve may be thought of as equivalent to theMann-Whitney U test, which tests for the median difference betweenscores obtained in the two groups considered if the groups are ofcontinuous data, or to the Wilcoxon test of ranks.

As discussed above, suitable tests may exhibit one or more of thefollowing results on these various measures: a specificity of greaterthan 0.5, preferably at least 0.6, more preferably at least 0.7, stillmore preferably at least 0.8, even more preferably at least 0.9 and mostpreferably at least 0.95, with a corresponding sensitivity greater than0.2, preferably greater than 0.3, more preferably greater than 0.4,still more preferably at least 0.5, even more preferably 0.6, yet morepreferably greater than 0.7, still more preferably greater than 0.8,more preferably greater than 0.9, and most preferably greater than 0.95;a sensitivity of greater than 0.5, preferably at least 0.6, morepreferably at least 0.7, still more preferably at least 0.8, even morepreferably at least 0.9 and most preferably at least 0.95, with acorresponding specificity greater than 0.2, preferably greater than 0.3,more preferably greater than 0.4, still more preferably at least 0.5,even more preferably 0.6, yet more preferably greater than 0.7, stillmore preferably greater than 0.8, more preferably greater than 0.9, andmost preferably greater than 0.95; at least 75% sensitivity, combinedwith at least 75% specificity; a ROC curve area of greater than 0.5,preferably at least 0.6, more preferably 0.7, still more preferably atleast 0.8, even more preferably at least 0.9, and most preferably atleast 0.95; an odds ratio different from 1, preferably at least about 2or more or about 0.5 or less, more preferably at least about 3 or moreor about 0.33 or less, still more preferably at least about 4 or more orabout 0.25 or less, even more preferably at least about 5 or more orabout 0.2 or less, and most preferably at least about 10 or more orabout 0.1 or less; a positive likelihood ratio (calculated assensitivity/(1-specificity)) of greater than 1, at least 2, morepreferably at least 3, still more preferably at least 5, and mostpreferably at least 10; and or a negative likelihood ratio (calculatedas (1-sensitivity)/specificity) of less than 1, less than or equal to0.5, more preferably less than or equal to 0.3, and most preferably lessthan or equal to 0.1

Additional clinical indicia may be combined with the appendicitisbiomarker assay result(s) of the present invention. Other clinicalindicia which may be combined with the appendicitis biomarker assayresult(s) of the present invention includes demographic information(e.g., weight, sex, age, race), medical history (e.g., family history,type of surgery, pre-existing disease such as aneurism, congestive heartfailure, preeclampsia, eclampsia, diabetes mellitus, hypertension,coronary artery disease, proteinuria, or renal insufficiency), riskscores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI,Framingham Risk Score), etc.

Combining assay results/clinical indicia in this manner can comprise theuse of multivariate logistical regression, loglinear modeling, neuralnetwork analysis, n-of-m analysis, decision tree analysis, etc. Thislist is not meant to be limiting.

Selecting a Treatment Regimen

Once a diagnosis is obtained, the clinician can readily select atreatment regimen that is compatible with the diagnosis. The skilledartisan is aware of appropriate treatments for numerous diseasesdiscussed in relation to the methods of diagnosis described herein. See,e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck ResearchLaboratories, Whitehouse Station, N.J., 1999. In addition, since themethods and compositions described herein provide prognosticinformation, the markers of the present invention may be used to monitora course of treatment. For example, improved or worsened prognosticstate may indicate that a particular treatment is or is not efficacious.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The examples providedherein are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention.

EXAMPLE 1. IMMUNOASSAY FORMAT

Analytes are measured using standard sandwich enzyme immunoassaytechniques. A first antibody which binds the analyte is immobilized inwells of a 96 well polystyrene microplate. Analyte standards and testsamples are pipetted into the appropriate wells and any analyte presentis bound by the immobilized antibody. After washing away any unboundsubstances, a horseradish peroxidase-conjugated second antibody whichbinds the analyte is added to the wells, thereby forming sandwichcomplexes with the analyte (if present) and the first antibody.Following a wash to remove any unbound antibody-enzyme reagent, asubstrate solution comprising tetramethylbenzidine and hydrogen peroxideis added to the wells. Color develops in proportion to the amount ofanalyte present in the sample. The color development is stopped and theintensity of the color is measured at 540 nm or 570 nm. An analyteconcentration is assigned to the test sample by comparison to a standardcurve determined from the analyte standards.

EXAMPLE 2. USE OF ANALYTE AS A MARKER FOR ASSESSING PATIENTS FORAPPENDICITIS

Patients from the emergency department (ED) classified as positive forappendicitis according to clinical diagnosis at the study site wereselected as a “diseased” cohort (“Cohort 1”). Plasma and urine samplesfrom each patient in Cohort 1 were collected at the time ofappendectomy. Cohort 2 was a normal population sampled separately. Theconcentrations of the analyte in these samples were measured by standardimmunoassay methods using commercially available assay reagents. Areceiver operating characteristic (ROC) curve was generated using theconcentrations, and the performance of the analyte is assessed by thearea under the ROC curve (AUC). The two-tailed p-value of the AUC forthe analyte was also calculated to determine statistical significance.“Inc/Dec” indicates if the marker is increasing or decreasing in Cohort1 relative to Cohort 2.

TABLE 1 Urine samples Increasing/ Preferred Name Units Decreasing pAdiponectin ng/ml inc 8.68E−07 Advanced glycosylation end pg/ml dec2.82E−01 product-specific receptor Alpha-2 macroglobulin ug/ml inc1.51E−04 Alpha-2-HS-glycoprotein ng/ml inc 1.97E−02 Angiopoietin-1 ng/mldec 7.54E−01 Antileukoproteinase pg/ml dec 9.51E−01 Apolipoprotein(a)ng/ml dec 6.85E−05 Carbonic anhydrase 9 ng/ml inc 3.80E−01 C-C motifchemokine 1 pg/ml inc 7.88E−01 C-C motif chemokine 13 pg/ml dec 3.19E−01C-C motif chemokine 15 pg/ml dec 1.45E−01 C-C motif chemokine 17 pg/mlinc 6.55E−01 C-C motif chemokine 19 pg/ml dec 5.62E−02 C-C motifchemokine 20 pg/ml dec 2.49E−01 C-C motif chemokine 21 pg/ml inc7.47E−01 C-C motif chemokine 22 pg/ml inc 7.81E−01 C-C motif chemokine23 ng/ml inc 5.63E−01 C-C motif chemokine 24 pg/ml inc 3.00E−02 C-Cmotif chemokine 26 pg/ml dec 6.44E−01 C-C motif chemokine 27 pg/ml inc3.18E−01 C-C motif chemokine 3 pg/ml dec 3.36E−01 C-C motif chemokine 4pg/ml inc 6.33E−01 C-C motif chemokine 7 pg/ml inc 6.44E−01 C-C motifchemokine 8 pg/ml inc 8.29E−01 Ceruloplasmin ng/ml dec 8.02E−02C-Peptide pg/ml dec 3.35E−04 Creatine Kinase-MB ng/ml inc 7.18E−01 C-X-Cmotif chemokine 10 pg/ml inc 6.59E−02 C-X-C motif chemokine 11 pg/ml inc6.35E−01 C-X-C motif chemokine 13 pg/ml dec 6.57E−01 C-X-C motifchemokine 16 ng/ml inc 4.61E−01 C-X-C motif chemokine 5 pg/ml inc8.20E−01 C-X-C motif chemokine 6 pg/ml inc 8.93E−06 C-X-C motifchemokine 9 pg/ml inc 2.25E−01 Cystatin-C ng/ml dec 2.01E−01 Endothelialprotein C receptor ng/ml inc 1.26E−02 Eotaxin pg/ml inc 7.99E−01Epidermal growth factor receptor pg/ml inc 7.11E−01 Ferritin pg/ml inc5.59E−06 Fibrinogen ng/ml inc 7.56E−01 Gastric inhibitory polypeptidepg/ml inc 6.44E−01 Glucagon pg/ml dec 8.18E−01 Glucagon-like peptide 1pg/ml inc 8.18E−01 Granulocyte-macrophage colony- pg/ml dec 9.87E−02stimulating factor Heparin-binding growth factor 2 pg/ml dec 7.31E−01Hepatocyte growth factor ng/ml dec 6.68E−01 Immunoglobulin A ng/ml inc6.83E−01 Immunoglobulin M ng/ml inc 2.64E−08 Immunoglogulin G1 ng/ml dec1.37E−02 Immunoglogulin G2 ng/ml dec 2.19E−01 Immunoglogulin G3 ng/mldec 7.25E−01 Immunoglogulin G4 ng/ml inc 4.56E−03 Insulin pg/ml dec4.31E−02 Insulin-like growth factor- ng/ml dec 2.00E−03 binding protein1 Insulin-like growth factor- ng/ml dec 0.00E+00 binding protein 2Insulin-like growth factor- ng/ml dec 1.96E−01 binding protein 3Insulin-like growth factor- ng/ml inc 8.52E−01 binding protein 4Insulin-like growth factor- ng/ml dec 6.96E−01 binding protein 5Insulin-like growth factor- ng/ml inc 1.17E−01 binding protein 6Insulin-like growth factor- ng/ml inc 2.36E−01 binding protein 7Interferon alpha-2 pg/ml dec 2.86E−01 Interferon gamma pg/ml inc8.31E−01 Interleukin-1 alpha pg/ml dec 5.10E−01 Interleukin-1 beta pg/mldec 9.67E−01 Interleukin-1 receptor pg/ml dec 4.62E−02 antagonistprotein Interleukin-1 receptor type I pg/mL inc 3.16E−01 Interleukin-1receptor type II pg/mL inc 9.27E−01 Interleukin-11 pg/ml dec 7.53E−03Interleukin-12 pg/ml inc 8.48E−01 Interleukin-12 subunit beta ng/ml dec5.34E−02 Interleukin-13 pg/ml dec 8.78E−01 Interleukin-15 pg/ml dec2.31E−01 Interleukin-2 pg/ml dec 5.87E−01 Interleukin-20 pg/ml dec3.22E−01 Interleukin-21 pg/ml dec 4.17E−01 Interleukin-23 pg/ml inc6.19E−01 Interleukin-28A pg/ml inc 4.52E−01 Interleukin-29 pg/ml inc3.74E−02 Interleukin-3 pg/ml inc 3.48E−01 Interleukin-33 pg/ml inc2.14E−01 Interleukin-4 pg/ml inc 5.71E−01 Interleukin-5 ng/ml inc8.08E−01 Interleukin-6 receptor subunit alpha pg/ml inc 6.70E−01Interleukin-6 receptor subunit beta pg/ml dec 6.63E−01 Interleukin-7pg/ml dec 8.59E−01 Interleukin-9 pg/ml inc 4.32E−01 Islet amyloidpolypeptide pg/ml dec 8.78E−01 Kit ligand pg/ml dec 3.64E−01 Leptinpg/ml dec 3.21E−01 Leukemia inhibitory factor pg/ml dec 8.27E−01Lymphotactin pg/ml inc 4.48E−01 Lymphotoxin-alpha pg/ml dec 6.44E−01Macrophage colony-stimulating pg/ml inc 1.01E−01 factor 1Metalloproteinase inhibitor 1 pg/ml inc 7.52E−01 Metalloproteinaseinhibitor 2 pg/ml inc 1.78E−01 Metalloproteinase inhibitor 3 pg/ml inc8.45E−01 Metalloproteinase inhibitor 4 pg/ml dec 1.62E−01 Mix ofGrowth-regulated alpha, pg/ml inc 4.65E−01 beta, and gamma proteinsMyoglobin ng/ml dec 1.14E−02 Pancreatic prohormone pg/ml dec 5.89E−01Peptide YY pg/ml dec 8.18E−01 Pro-epidermal growth factor pg/ml inc5.02E−02 Pro-interleukin-16 pg/ml dec 9.39E−01 Protein S100-A12 ng/mldec 2.49E−02 Protransforming growth factor alpha pg/ml inc 5.62E−02Secretory immunoglobulin A ng/ml inc 9.18E−01 Serum amyloid P-componentng/ml inc 1.02E−04 SL cytokine pg/ml dec 7.11E−01 Stromal cell-derivedfactor 1 pg/ml dec 7.03E−02 Thrombopoietin pg/ml dec 1.15E−02 Thymicstromal lymphopoietin pg/ml inc 1.81E−03 Tumor necrosis factor pg/ml dec8.29E−01 Tumor necrosis factor ligand pg/ml inc 1.24E−01 superfamilymember 10 Tumor necrosis factor receptor pg/ml dec 5.55E−03 superfamilymember 1A Tumor necrosis factor receptor pg/ml dec 4.11E−02 superfamilymember 1B Vascular endothelial growth factor pg/ml dec 1.11E−01 receptor1 Vascular endothelial growth factor pg/ml dec 6.61E−01 receptor 2Vascular endothelial growth factor pg/ml dec 9.11E−01 receptor 3 vonWillebrand Factor ng/ml inc 2.52E−04

TABLE 2 Plasma samples Increasing/ Preferred Name Units Decreasing p 72kDa type IV collagenase pg/ml inc 1.73E−14 Adiponectin ng/ml inc8.92E−01 Advanced glycosylation end product-specific receptor pg/ml inc3.24E−01 Alpha-2 macroglobulin ug/ml dec 1.52E−01Alpha-2-HS-glycoprotein ng/ml inc 4.26E−04 Alpha-fetoprotein pg/ml inc8.85E−01 Angiopoietin-1 ng/ml inc 1.75E−02 Antileukoproteinase pg/ml dec3.89E−01 Apolipoprotein(a) ng/ml inc 3.51E−01 Cancer Antigen 15-3 U/mlinc 9.18E−06 Cancer Antigen 19-9 U/ml inc 6.13E−01 Carbonic anhydrase 9ng/ml inc 3.78E−02 Carcinoembryonic antigen-related cell adhesionmolecule 5 pg/ml dec 9.44E−01 C-C motif chemokine 1 pg/ml dec 1.79E−01C-C motif chemokine 13 pg/ml inc 5.89E−03 C-C motif chemokine 15 pg/mldec 1.67E−01 C-C motif chemokine 17 pg/ml inc 1.69E−01 C-C motifchemokine 19 pg/ml inc 2.24E−03 C-C motif chemokine 20 pg/ml dec4.43E−01 C-C motif chemokine 21 pg/ml dec 1.43E−02 C-C motif chemokine22 pg/ml dec 3.28E−06 C-C motif chemokine 23 ng/ml inc 1.25E−08 C-Cmotif chemokine 24 pg/ml inc 3.32E−01 C-C motif chemokine 26 pg/ml inc1.54E−01 C-C motif chemokine 27 pg/ml dec 1.44E−02 C-C motif chemokine 3pg/ml dec 1.81E−01 C-C motif chemokine 4 pg/ml inc 3.44E−04 C-C motifchemokine 7 pg/ml inc 8.34E−01 C-C motif chemokine 8 pg/ml dec 9.23E−01Ceruloplasmin ng/ml dec 9.62E−02 Choriogonadotropin subunit beta mU/mlinc 2.52E−02 Collagenase 3 pg/ml dec 4.64E−01 C-Peptide pg/ml dec2.52E−05 Creatine Kinase-MB ng/ml inc 3.37E−01 C-X-C motif chemokine 10pg/ml inc 1.08E−01 C-X-C motif chemokine 11 pg/ml dec 6.95E−02 C-X-Cmotif chemokine 13 pg/ml dec 5.59E−03 C-X-C motif chemokine 16 ng/ml dec1.47E−02 C-X-C motif chemokine 5 pg/ml dec 9.76E−01 C-X-C motifchemokine 6 pg/ml inc 1.04E−01 C-X-C motif chemokine 9 pg/ml inc4.93E−01 Cystatin-C ng/ml inc 8.02E−07 Endothelial protein C receptorng/ml dec 3.19E−01 Eotaxin pg/ml inc 1.20E−07 Epidermal growth factorreceptor pg/ml dec 6.80E−01 Ferritin pg/ml dec 1.92E−04 Fibrinogen ug/mldec 5.85E−02 Gastric inhibitory polypeptide pg/ml dec 0.00E+00 Glucagonpg/ml dec 8.82E−01 Glucagon-like peptide 1 pg/ml dec 6.75E−04Granulocyte-macrophage colony-stimulating factor pg/ml inc 2.07E−02Heparin-binding growth factor 2 pg/ml inc 7.22E−01 Hepatocyte growthfactor ng/ml inc 0.00E+00 Immunoglobulin A ng/ml dec 4.10E−03Immunoglobulin M ng/ml dec 5.79E−01 Immunoglogulin G1 ng/ml dec 4.93E−01Immunoglogulin G2 ng/ml dec 3.03E−01 Immunoglogulin G3 ng/ml dec2.08E−04 Immunoglogulin G4 ng/ml dec 4.64E−01 Insulin pg/ml dec 8.32E−05Insulin-like growth factor-binding protein 1 ng/ml dec 2.19E−10Insulin-like growth factor-binding protein 2 ng/ml dec 8.69E−01Insulin-like growth factor-binding protein 3 ng/ml inc 1.75E−01Insulin-like growth factor-binding protein 4 ng/ml dec 5.07E−01Insulin-like growth factor-binding protein 5 ng/ml dec 9.25E−04Insulin-like growth factor-binding protein 6 ng/ml inc 1.68E−07Insulin-like growth factor-binding protein 7 ng/ml dec 7.07E−05Interferon alpha-2 pg/ml inc 1.55E−01 Interferon gamma pg/ml inc1.04E−02 Interleukin-1 alpha pg/ml dec 8.53E−01 Interleukin-1 beta pg/mldec 1.45E−01 Interleukin-1 receptor type I pg/mL dec 1.60E−03Interleukin-1 receptor type II pg/mL inc 7.29E−01 Interleukin-11 pg/mlinc 5.15E−01 Interleukin-12 pg/ml inc 5.94E−01 Interleukin-12 subunitbeta ng/ml dec 9.09E−01 Interleukin-13 pg/ml inc 4.61E−01 Interleukin-15pg/ml dec 7.13E−02 Interleukin-2 pg/ml dec 1.90E−03 Interleukin-20 pg/mlinc 5.51E−01 Interleukin-21 pg/ml dec 9.01E−01 Interleukin-23 pg/ml dec5.67E−01 Interleukin-28A pg/ml dec 4.44E−01 Interleukin-29 pg/ml dec3.04E−01 Interleukin-3 pg/ml inc 6.55E−01 Interleukin-33 pg/ml dec5.24E−01 Interleukin-4 pg/ml dec 4.79E−01 Interleukin-5 ng/ml inc2.65E−01 Interleukin-6 receptor subunit alpha pg/ml dec 2.88E−01Interleukin-6 receptor subunit beta pg/ml dec 5.47E−01 Interleukin-7pg/ml inc 7.57E−01 Interleukin-9 pg/ml dec 5.16E−01 Interstitialcollagenase pg/ml dec 1.78E−05 Islet amyloid polypeptide pg/ml inc2.96E−07 Keratin, type I cytoskeletal 19 (aa311-367) pg/ml dec 9.39E−01Kit ligand pg/ml inc 8.87E−01 Leptin pg/ml dec 8.44E−01 Leukemiainhibitory factor pg/ml dec 3.49E−01 Lymphotactin pg/ml dec 5.21E−02Lymphotoxin-alpha pg/ml inc 6.48E−01 Macrophage colony-stimulatingfactor 1 pg/ml dec 7.41E−04 Macrophage metalloelastase pg/ml inc8.74E−01 Macrophage migration inhibitory factor pg/ml inc 5.75E−06Matrilysin pg/ml dec 1.59E−01 Metalloproteinase inhibitor 1 pg/ml dec9.56E−01 Metalloproteinase inhibitor 2 pg/ml inc 4.10E−09Metalloproteinase inhibitor 3 pg/ml inc 2.28E−01 Metalloproteinaseinhibitor 4 pg/ml inc 4.21E−01 Mix of Growth-regulated alpha, beta, andgamma proteins pg/ml inc 2.33E−01 Myoglobin ng/ml dec 5.72E−01Pancreatic prohormone pg/ml inc 2.44E−13 Peptide YY pg/ml inc 1.32E−03Pro-epidermal growth factor pg/ml inc 1.23E−02 Pro-interleukin-16 pg/mlinc 3.21E−01 Prolactin pg/ml dec 2.55E−11 Prostate-specific antigenpg/ml dec 7.49E−01 Protein S100-A12 ng/ml dec 2.02E−04 Protransforminggrowth factor alpha pg/ml inc 3.50E−02 Serum amyloid P-component ng/mldec 4.77E−01 SL cytokine pg/ml dec 1.27E−01 Stromal cell-derived factor1 pg/ml inc 8.60E−01 Stromelysin-1 pg/ml dec 2.76E−06 Thrombopoietinpg/ml inc 1.76E−04 Thymic stromal lymphopoietin pg/ml dec 5.75E−01 Tumornecrosis factor pg/ml dec 1.15E−01 Tumor necrosis factor ligandsuperfamily member 10 pg/ml inc 1.03E−06 Tumor necrosis factor ligandsuperfamily member 6 pg/ml dec 3.96E−01 Tumor necrosis factor receptorsuperfamily member pg/ml dec 1.20E−07 1A Tumor necrosis factor receptorsuperfamily member pg/ml dec 1.03E−02 1B Vascular endothelial growthfactor receptor 1 pg/ml inc 6.04E−07 Vascular endothelial growth factorreceptor 2 pg/ml dec 2.63E−03 Vascular endothelial growth factorreceptor 3 pg/ml inc 8.51E−01 von Willebrand Factor ng/ml dec 8.01E−01WAP four-disulfide core domain protein 2 pg/ml dec 4.13E−04

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention. The examplesprovided herein are representative of preferred embodiments, areexemplary, and are not intended as limitations on the scope of theinvention. Modifications therein and other uses will occur to thoseskilled in the art. These modifications are encompassed within thespirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a person skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe invention pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof” and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments and optional features, modification and variationof the concepts herein disclosed may be resorted to by those skilled inthe art, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

Other embodiments are set forth within the following claims.

We claim:
 1. A method of diagnosing appendicitis in a subject, orassigning a likelihood of a future outcome to a subject diagnosed withappendicitis, comprising: performing one or more assays configured todetect one or more biomarkers selected from the group consisting of 72kDa type IV collagenase, Adiponectin, Advanced glycosylation endproduct-specific receptor, Alpha-2 macroglobulin,Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1,Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, CancerAntigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-relatedcell adhesion molecule 5, C—C motif chemokine 1, C—C motif chemokine 13,C—C motif chemokine 15, C—C motif chemokine 17, C—C motif chemokine 19,C—C motif chemokine 20, C—C motif chemokine 21, C—C motif chemokine 22,C—C motif chemokine 23, C—C motif chemokine 24, C—C motif chemokine 26,C—C motif chemokine 27, C—C motif chemokine 3, C—C motif chemokine 4,C—C motif chemokine 7, C—C motif chemokine 8, Ceruloplasmin,Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, CreatineKinase-MB, C—X—C motif chemokine 10, C—X—C motif chemokine 11, C—X—Cmotif chemokine 13, C—X—C motif chemokine 16, C—X—C motif chemokine 5,C—X—C motif chemokine 6, C—X—C motif chemokine 9, Cystatin-C,Endothelial protein C receptor, Eotaxin, Epidermal growth factorreceptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide,Glucagon, Glucagon-like peptide 1, Granulocyte-macrophagecolony-stimulating factor, Growth-regulated alpha, beta, and gammaproteins, Heparin-binding growth factor 2, Hepatocyte growth factor,Immuno globulin A, Immunoglobulin M, Immunoglogulin G1, ImmunoglogulinG2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growthfactor-binding protein 1, Insulin-like growth factor-binding protein 2,Insulin-like growth factor-binding protein 3, Insulin-like growthfactor-binding protein 4, Insulin-like growth factor-binding protein 5,Insulin-like growth factor-binding protein 6, Insulin-like growthfactor-binding protein 7, Interferon alpha-2, Interferon gamma,Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptorantagonist protein, Interleukin-1 receptor type I, Interleukin-1receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunitbeta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20,Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29,Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5,Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunitbeta, Interleukin-7, Interleukin-9, Interstitial collagenase, Isletamyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kitligand, Leptin, Leukemia inhibitory factor, Lymphotactin,Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophagemetalloelastase, Macrophage migration inhibitory factor, Matrilysin,Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2,Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin,Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor,Pro-interleukin-16, Prolactin, Prostate-specific antigen, ProteinS100-A12, Protransforming growth factor alpha, Secretory immunoglobulinA, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumornecrosis factor, Tumor necrosis factor ligand superfamily member 10,Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factorreceptor superfamily member 1A, Tumor necrosis factor receptorsuperfamily member 1B, Vascular endothelial growth factor receptor 1,Vascular endothelial growth factor receptor 2, Vascular endothelialgrowth factor receptor 3, von Willebrand Factor, and WAP four-disulfidecore domain protein 2 on a body fluid sample obtained from the subjectto provide one or more assay result(s); and correlating the assayresult(s) to the occurrence or nonoccurrence of appendicitis in thesubject or likelihood of the future outcome to the subject.
 2. A methodaccording to claim 1, wherein the performing step comprises introducingthe body fluid sample obtained from the subject into an assay instrumentwhich (i) contacts the body fluid sample with one or more bindingreagents corresponding to the biomarker(s) being assayed, wherein eachbiomarker which is assayed binds to its respective specific bindingreagent in an amount related to its concentration in the body fluidsample, (ii) generates one or more assay results indicative of bindingof each biomarker which is assayed to its respective specific bindingreagent; and (iii) displays the one or more assay results as aquantitative result in a human-readable form.
 3. A method according toclaim 1 or 2, wherein the future outcome is mortality.
 4. A methodaccording to claim 1 or 2, wherein the subject is being evaluated forabdominal pain.
 5. A method according to claim 1 or 2, wherein thecorrelating step comprises determining the concentration of eachbiomarker which is assayed, and individually comparing each biomarkerconcentration to a corresponding threshold level for that biomarker. 6.A method according to claim 5, wherein the assay instrument performs thecorrelating step, which comprises determining the concentration of eachbiomarker which is assayed, individually comparing each biomarkerconcentration to a corresponding threshold level for that biomarker, anddisplaying an indication of whether each biomarker does or does notexceed its corresponding threshold in a human-readable form.
 7. A methodaccording to claim 2, wherein a plurality of the biomarkers aremeasured, wherein the assay instrument performs the correlating step,which comprises determining the concentration of each of the pluralityof biomarkers, calculating a single value based on the concentration ofeach of the plurality of biomarkers, comparing the single value to acorresponding threshold level and displaying an indication of whetherthe single value does or does not exceed its corresponding threshold ina human-readable form.
 8. A method according to one of claims 1-7,wherein method provides a sensitivity or specificity of at least 0.7 forthe identification of appendicitis when compared to normal subjects. 9.A method according to one of claims 1-7, wherein method provides asensitivity or specificity of at least 0.7 for the identification ofappendicitis when compared to subjects exhibiting symptoms that mimicappendicitis symptoms.
 10. A method according to one of claims 1-9,wherein the sample is selected from the group consisting of blood,serum, and plasma.
 11. A method for evaluating biomarker levels in abody fluid sample, comprising: obtaining a body fluid sample from asubject selected for evaluation based on a determination that thesubject is experiencing symptoms indicative of possible acuteappendicitis; and performing one or more analyte binding assaysconfigured to detect one or more biomarkers selected from the groupconsisting of 72 kDa type IV collagenase, Adiponectin, Advancedglycosylation end product-specific receptor, Alpha-2 macroglobulin,Alpha-2-HS-glycoprotein, Alpha-fetoprotein, Angiopoietin-1,Antileukoproteinase, Apolipoprotein(a), Cancer Antigen 15-3, CancerAntigen 19-9, Carbonic anhydrase 9, Carcinoembryonic antigen-relatedcell adhesion molecule 5, C—C motif chemokine 1, C—C motif chemokine 13,C—C motif chemokine 15, C—C motif chemokine 17, C—C motif chemokine 19,C—C motif chemokine 20, C—C motif chemokine 21, C—C motif chemokine 22,C—C motif chemokine 23, C—C motif chemokine 24, C—C motif chemokine 26,C—C motif chemokine 27, C—C motif chemokine 3, C—C motif chemokine 4,C—C motif chemokine 7, C—C motif chemokine 8, Ceruloplasmin,Choriogonadotropin subunit beta, Collagenase 3, C-Peptide, CreatineKinase-MB, C—X—C motif chemokine 10, C—X—C motif chemokine 11, C—X—Cmotif chemokine 13, C—X—C motif chemokine 16, C—X—C motif chemokine 5,C—X—C motif chemokine 6, C—X—C motif chemokine 9, Cystatin-C,Endothelial protein C receptor, Eotaxin, Epidermal growth factorreceptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide,Glucagon, Glucagon-like peptide 1, Granulocyte-macrophagecolony-stimulating factor, Growth-regulated alpha, beta, and gammaproteins, Heparin-binding growth factor 2, Hepatocyte growth factor,Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, ImmunoglogulinG2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growthfactor-binding protein 1, Insulin-like growth factor-binding protein 2,Insulin-like growth factor-binding protein 3, Insulin-like growthfactor-binding protein 4, Insulin-like growth factor-binding protein 5,Insulin-like growth factor-binding protein 6, Insulin-like growthfactor-binding protein 7, Interferon alpha-2, Interferon gamma,Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptorantagonist protein, Interleukin-1 receptor type I, Interleukin-1receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunitbeta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20,Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29,Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5,Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunitbeta, Interleukin-7, Interleukin-9, Interstitial collagenase, Isletamyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kitligand, Leptin, Leukemia inhibitory factor, Lymphotactin,Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophagemetalloelastase, Macrophage migration inhibitory factor, Matrilysin,Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2,Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin,Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor,Pro-interleukin-16, Prolactin, Prostate-specific antigen, ProteinS100-A12, Protransforming growth factor alpha, Secretory immunoglobulinA, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumornecrosis factor, Tumor necrosis factor ligand superfamily member 10,Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factorreceptor superfamily member 1A, Tumor necrosis factor receptorsuperfamily member 1B, Vascular endothelial growth factor receptor 1,Vascular endothelial growth factor receptor 2, Vascular endothelialgrowth factor receptor 3, von Willebrand Factor, and WAP four-disulfidecore domain protein 2 by introducing the body fluid sample obtained fromthe subject into an assay instrument which (i) contacts the body fluidsample with one or more binding reagents corresponding to thebiomarker(s) being assayed, wherein each biomarker which is assayedbinds to its respective specific binding reagent in an amount related toits concentration in the body fluid sample, (ii) generates one or moreassay results indicative of binding of each biomarker which is assayedto its respective specific binding reagent; and (iii) displays the oneor more assay results as a quantitative result in a human-readable form.12. A method according to claim 11, wherein the assay result(s) aredisplayed as a concentration of each biomarker which is assayed.
 13. Amethod according to claim 12, wherein the assay instrument furtherindividually compares each biomarker concentration to a correspondingthreshold level for that biomarker, and displays an indication ofwhether each biomarker does or does not exceed its correspondingthreshold in a human-readable form.
 14. A method according to claim 11,wherein a plurality of the biomarkers are measured, and wherein theassay results(s) comprise a single value calculated using a functionthat converts the concentration of each of the plurality of biomarkersinto a single value.
 15. A method according to claim 14, wherein theassay instrument further compares the single value to a correspondingthreshold level and displays an indication of whether the single valuedoes or does not exceed its corresponding threshold in a human-readableform.
 16. A method for evaluating biomarker levels in a body fluidsample, comprising: obtaining a body fluid sample from a subjectselected for evaluation based on a determination that the subject hasbeen diagnosed with acute appendicitis; and performing one or moreanalyte binding assays configured to detect one or more biomarkersselected from the group consisting of 72 kDa type IV collagenase,Adiponectin, Advanced glycosylation end product-specific receptor,Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein, Alpha-fetoprotein,Angiopoietin-1, Antileukoproteinase, Apolipoprotein(a), Cancer Antigen15-3, Cancer Antigen 19-9, Carbonic anhydrase 9, Carcinoembryonicantigen-related cell adhesion molecule 5, C—C motif chemokine 1, C—Cmotif chemokine 13, C—C motif chemokine 15, C—C motif chemokine 17, C—Cmotif chemokine 19, C—C motif chemokine 20, C—C motif chemokine 21, C—Cmotif chemokine 22, C—C motif chemokine 23, C—C motif chemokine 24, C—Cmotif chemokine 26, C—C motif chemokine 27, C—C motif chemokine 3, C—Cmotif chemokine 4, C—C motif chemokine 7, C—C motif chemokine 8,Ceruloplasmin, Choriogonadotropin subunit beta, Collagenase 3,C-Peptide, Creatine Kinase-MB, C—X—C motif chemokine 10, C—X—C motifchemokine 11, C—X—C motif chemokine 13, C—X—C motif chemokine 16, C—X—Cmotif chemokine 5, C—X—C motif chemokine 6, C—X—C motif chemokine 9,Cystatin-C, Endothelial protein C receptor, Eotaxin, Epidermal growthfactor receptor, Ferritin, Fibrinogen, Gastric inhibitory polypeptide,Glucagon, Glucagon-like peptide 1, Granulocyte-macrophagecolony-stimulating factor, Growth-regulated alpha, beta, and gammaproteins, Heparin-binding growth factor 2, Hepatocyte growth factor,Immunoglobulin A, Immunoglobulin M, Immunoglogulin G1, ImmunoglogulinG2, Immunoglogulin G3, Immunoglogulin G4, Insulin, Insulin-like growthfactor-binding protein 1, Insulin-like growth factor-binding protein 2,Insulin-like growth factor-binding protein 3, Insulin-like growthfactor-binding protein 4, Insulin-like growth factor-binding protein 5,Insulin-like growth factor-binding protein 6, Insulin-like growthfactor-binding protein 7, Interferon alpha-2, Interferon gamma,Interleukin-1 alpha, Interleukin-1 beta, Interleukin-1 receptorantagonist protein, Interleukin-1 receptor type I, Interleukin-1receptor type II, Interleukin-11, Interleukin-12, Interleukin-12 subunitbeta, Interleukin-13, Interleukin-15, Interleukin-2, Interleukin-20,Interleukin-21, Interleukin-23, Interleukin-28A, Interleukin-29,Interleukin-3, Interleukin-33, Interleukin-4, Interleukin-5,Interleukin-6 receptor subunit alpha, Interleukin-6 receptor subunitbeta, Interleukin-7, Interleukin-9, Interstitial collagenase, Isletamyloid polypeptide, Keratin, type I cytoskeletal 19 (aa311-367), Kitligand, Leptin, Leukemia inhibitory factor, Lymphotactin,Lymphotoxin-alpha, Macrophage colony-stimulating factor 1, Macrophagemetalloelastase, Macrophage migration inhibitory factor, Matrilysin,Metalloproteinase inhibitor 1, Metalloproteinase inhibitor 2,Metalloproteinase inhibitor 3, Metalloproteinase inhibitor 4, Myoglobin,Pancreatic prohormone, Peptide YY, Pro-epidermal growth factor,Pro-interleukin-16, Prolactin, Prostate-specific antigen, ProteinS100-A12, Protransforming growth factor alpha, Secretory immunoglobulinA, Serum amyloid P-component, SL cytokine, Stromal cell-derived factor1, Stromelysin-1, Thrombopoietin, Thymic stromal lymphopoietin, Tumornecrosis factor, Tumor necrosis factor ligand superfamily member 10,Tumor necrosis factor ligand superfamily member 6, Tumor necrosis factorreceptor superfamily member 1A, Tumor necrosis factor receptorsuperfamily member 1B, Vascular endothelial growth factor receptor 1,Vascular endothelial growth factor receptor 2, Vascular endothelialgrowth factor receptor 3, von Willebrand Factor, and WAP four-disulfidecore domain protein 2 by introducing the body fluid sample obtained fromthe subject into an assay instrument which (i) contacts the body fluidsample with one or more binding reagents corresponding to thebiomarker(s) being assayed, wherein each biomarker which is assayedbinds to its respective specific binding reagent in an amount related toits concentration in the body fluid sample, (ii) generates one or moreassay results indicative of binding of each biomarker which is assayedto its respective specific binding reagent; and (iii) displays the oneor more assay results as a quantitative result in a human-readable form.17. A method according to claim 16, wherein the assay result(s) aredisplayed as a concentration of each biomarker which is assayed.
 18. Amethod according to claim 17, wherein the assay instrument furtherindividually compares each biomarker concentration to a correspondingthreshold level for that biomarker, and displays an indication ofwhether each biomarker does or does not exceed its correspondingthreshold in a human-readable form.
 19. A method according to claim 16,wherein a plurality of the biomarkers are measured, and wherein theassay results(s) comprise a single value calculated using a functionthat converts the concentration of each of the plurality of biomarkersinto a single value.
 20. A method according to claim 19, wherein theassay instrument further compares the single value to a correspondingthreshold level and displays an indication of whether the single valuedoes or does not exceed its corresponding threshold in a human-readableform.
 21. A method according to one of claims 16-19, wherein the subjectis selected for evaluation of a mortality risk within a period selectedfrom the group consisting of 21 days, 14 days, 7 days, 5 days, 96 hours,72 hours, 48 hours, 36 hours, 24 hours, and 12 hours.
 22. A methodaccording to one of claims 11-21, wherein the plurality of assays areimmunoassays performed by (i) introducing the body fluid sample into anassay device comprising a plurality of antibodies, at least one of whichbinds to each biomarker which is assayed, and (ii) generating an assayresult indicative of binding of each biomarker to its respectiveantibody.
 23. A system for evaluating biomarker levels, comprising: aplurality of reagents which specifically bind for detection a pluralityof biomarkers selected from the group consisting of 72 kDa type IVcollagenase, Adiponectin, Advanced glycosylation end product-specificreceptor, Alpha-2 macroglobulin, Alpha-2-HS-glycoprotein,Alpha-fetoprotein, Angiopoietin-1, Antileukoproteinase,Apolipoprotein(a), Cancer Antigen 15-3, Cancer Antigen 19-9, Carbonicanhydrase 9, Carcinoembryonic antigen-related cell adhesion molecule 5,C—C motif chemokine 1, C—C motif chemokine 13, C—C motif chemokine 15,C—C motif chemokine 17, C—C motif chemokine 19, C—C motif chemokine 20,C—C motif chemokine 21, C—C motif chemokine 22, C—C motif chemokine 23,C—C motif chemokine 24, C—C motif chemokine 26, C—C motif chemokine 27,C—C motif chemokine 3, C—C motif chemokine 4, C—C motif chemokine 7, C—Cmotif chemokine 8, Ceruloplasmin, Choriogonadotropin subunit beta,Collagenase 3, C-Peptide, Creatine Kinase-MB, C—X—C motif chemokine 10,C—X—C motif chemokine 11, C—X—C motif chemokine 13, C—X—C motifchemokine 16, C—X—C motif chemokine 5, C—X—C motif chemokine 6, C—X—Cmotif chemokine 9, Cystatin-C, Endothelial protein C receptor, Eotaxin,Epidermal growth factor receptor, Ferritin, Fibrinogen, Gastricinhibitory polypeptide, Glucagon, Glucagon-like peptide 1,Granulocyte-macrophage colony-stimulating factor, Growth-regulatedalpha, beta, and gamma proteins, Heparin-binding growth factor 2,Hepatocyte growth factor, Immunoglobulin A, Immunoglobulin M,Immunoglogulin G1, Immunoglogulin G2, Immunoglogulin G3, ImmunoglogulinG4, Insulin, Insulin-like growth factor-binding protein 1, Insulin-likegrowth factor-binding protein 2, Insulin-like growth factor-bindingprotein 3, Insulin-like growth factor-binding protein 4, Insulin-likegrowth factor-binding protein 5, Insulin-like growth factor-bindingprotein 6, Insulin-like growth factor-binding protein 7, Interferonalpha-2, Interferon gamma, Interleukin-1 alpha, Interleukin-1 beta,Interleukin-1 receptor antagonist protein, Interleukin-1 receptor typeI, Interleukin-1 receptor type II, Interleukin-11, Interleukin-12,Interleukin-12 subunit beta, Interleukin-13, Interleukin-15,Interleukin-2, Interleukin-20, Interleukin-21, Interleukin-23,Interleukin-28A, Interleukin-29, Interleukin-3, Interleukin-33,Interleukin-4, Interleukin-5, Interleukin-6 receptor subunit alpha,Interleukin-6 receptor subunit beta, Interleukin-7, Interleukin-9,Interstitial collagenase, Islet amyloid polypeptide, Keratin, type Icytoskeletal 19 (aa311-367), Kit ligand, Leptin, Leukemia inhibitoryfactor, Lymphotactin, Lymphotoxin-alpha, Macrophage colony-stimulatingfactor 1, Macrophage metalloelastase, Macrophage migration inhibitoryfactor, Matrilysin, Metalloproteinase inhibitor 1, Metalloproteinaseinhibitor 2, Metalloproteinase inhibitor 3, Metalloproteinase inhibitor4, Myoglobin, Pancreatic prohormone, Peptide YY, Pro-epidermal growthfactor, Pro-interleukin-16, Prolactin, Prostate-specific antigen,Protein S100-A12, Protransforming growth factor alpha, Secretoryimmunoglobulin A, Serum amyloid P-component, SL cytokine, Stromalcell-derived factor 1, Stromelysin-1, Thrombopoietin, Thymic stromallymphopoietin, Tumor necrosis factor, Tumor necrosis factor ligandsuperfamily member 10, Tumor necrosis factor ligand superfamily member6, Tumor necrosis factor receptor superfamily member 1A, Tumor necrosisfactor receptor superfamily member 1B, Vascular endothelial growthfactor receptor 1, Vascular endothelial growth factor receptor 2,Vascular endothelial growth factor receptor 3, von Willebrand Factor,and WAP four-disulfide core domain protein 2; an assay instrumentconfigured to (i) receive a body fluid sample, (ii) contact theplurality of reagents with the body fluid sample and (iii) generate andquantitatively display in human readable form one or more assay resultsindicative of binding of each biomarker which is assayed to a respectivespecific binding reagent in the plurality of reagents.
 24. A systemaccording to claim 23 wherein the reagents comprise a plurality ofantibodies, at least one of which binds to each of the biomarkers whichare assayed.
 25. A system according to claim 24 wherein assay instrumentcomprises an assay device and an assay device reader, wherein theplurality of antibodies are immobilized at a plurality of predeterminedlocations within the assay device, wherein the assay device isconfigured to receive the body fluid sample such that the body fluidsample contacts the plurality of predetermined locations, and whereinthe assay device reader interrogates the plurality of predeterminedlocations to generate the assay results.